The leukocyte NADPH oxidase subunit p47PHOX: the role of the cysteine residues.
The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47PHOX, one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47PHOX-deficient Epstein-Barr virus-transformed B lymphoblasts, and O2- production by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O2- production by C196A was significantly larger than seen with wild type. The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O2- production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity.
Inanami
O
O
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
Johnson
J L
JL
Babior
B M
BM
D001402:B-Lymphocytes / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism*; D017382:Reactive Oxygen Species
D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D003545:Cysteine